Assay for detection of human parvovirus B19 nucleic acid

ABSTRACT

Nucleic acid oligomers specific for human parvovirus B19 genomic DNA are disclosed. An assay for amplifying and detecting human parvovirus B19 nucleic acid in biological specimens is disclosed. Compositions for detecting the presence of parvovirus B19 genomic DNA in human biological specimens are disclosed.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/316,691, filed Aug. 31, 2001, under 35 U.S.C. 119(e).

FIELD OF THE INVENTION

[0002] This invention relates to diagnostic methods and compositions fordetecting a human infectious agent, and specifically relates to methodsand compositions for detecting the nucleic acid of parvovirus B19 invitro.

BACKGROUND OF THE INVENTION

[0003] Human parvovirus B19 (sometimes called erythrovirus) is a bloodborne, non-enveloped virus that has a single-stranded DNA (ssDNA) genomeof about 5.5 kb (Shade et al., 1986, J. Virol. 58(3): 921-936, Brown etal., 1997, Ann. Rev. Med. 48: 59-67). Individual virions contain onecopy of either the plus or minus strand of the genome, represented inapproximately equal numbers. The ssDNA genome has inverted terminalrepeats that form 5′ and 3′ hairpins of about 350 nt which are essentialfor viral replication. The genome includes two open reading frames onthe plus strand which code for structural proteins (VP1 and VP2) andnon-structural protein (NS1).

[0004] Infection with parvovirus B19 can occur via respiratorytransmission or through infected blood or blood products. Viremia canreach high levels (e.g., up to 1011 per ml of blood) at about a weekafter inoculation, but is generally cleared within about two weeksfollowing infection. Infected individuals may exhibit no symptoms, orhave erythema infectiosum symptoms that include mild flu-like symptoms,rash, and/or temporary arthritis-like joint pain (arthropathy). Childrenare more likely than adults to develop the rash (called “fifthdisease”), whereas arthropathy is a common symptom in adults. Moreserious problems occur in susceptible patients, including aplasticcrisis in patients with hemolytic anemias, and persistent parvovirusinfection and other hematologic changes in immunosuppressed patients. Inwomen, parvovirus B19 infections have been associated with loss of about10% of early pregnancies due to fetal death.

[0005] Parvovirus B19 is a relatively resistant to viral inactivation,e.g., by chemical or heat-treatment methods used to destroy infectiveparticles in blood, serum or plasma. Also, high viral concentrations ina sample may overwhelm viral depletion methods used to remove viralcontaminants from the sample. Parvovirus B19 in blood, plasma orplasma-derived products can infect additional individuals who receivecontaminated transfusions or products. Plasma derivatives are often madefrom pooled donations (e.g., a pool of thousands of individualdonations) resulting in the risk that a single contaminated donationcould contaminate the pool and products derived from it. Thus, there isa need to detect the presence of human parvovirus B19 in biologicalsamples, such as donated blood or plasma to prevent further infection.There is also a need for an assay that detects parvovirus with asensitivity that allows detection of low titres of virus as may occurearly in an infection or in diluted or pooled samples. An assay forparvovirus B19 nucleic acid which detects an appropriate level ofcontamination may facilitate removal of infected donated units from theblood supply or contaminated lots of pooled plasma before use.

[0006] Immunodiagnostic methods have been used to identify blood, serumor plasma that is potentially contaminated with parvovirus B19. Manymethods detect anti-parvovirus antibodies (IgM or IgG) present in anindividual's serum or plasma (e.g., see PCT Nos. WO 96/09391 by Wolf etal. and WO 96/27799 by Hedman et al.). Immunological methods, however,have limitations on detecting recent or current infections because theyrely on detecting the body's response to the infectious agent. Becauseof the rapid rise in viremia following infection, an individual's bloodmay contain high levels of parvovirus B19 before anti-parvovirusantibodies are detectable, leading to false negative results. Becauseviremia is often quickly cleared, a person may remain antibody-positiveeven when infective particles are not present, leading to false positiveresults. Also, up to about 90% of adults are seropositive for parvovirusB19, making accurate immunological detection of recent or currentinfections difficult. Other assays detect the presence of parvovirus B19by detecting the virus or empty viral capsid bound to a purifiedcellular receptor (U.S. Pat. No. 5,449,608 to Young et al.).

[0007] DNA hybridization and amplification methods have been used todetect human parvovirus B19. U.S. Pat. No. 5,688,669 to Murtagh et al.describes detection of parvovirus B19 by amplifying a 284 bp portion ofparvovirus B19 DNA by using PCR and then digesting the amplified dsDNAwith exonuclease to make ssDNA. The ssDNA was hybridized with twoseparate oligonucleotide probes, a capture probe and a detection probe,and hybrids were detected on a solid support, e.g., by using acolorimetric assay in a microtiter plate. U.S. Pat. No. 6,183,999 toWeimer et al. discloses a nucleic acid amplification assay to detecthigh-titer parvovirus B19 in plasma protein solutions. By amplifying DNAin the NS1 gene using suboptimal annealing and elongation temperatures,the assay is used to indicate the presence of exceptionally largeamounts of pathogenic viruses in the sample. PCT No. WO 96/09391 by Wolfet al. describes cloning of the sequence that encodes the NS-1 proteinby linking PCR amplified fragments. Nested PCR and/or dot-blot assayswere used to detect parvovirus DNA in patients' sera, and results werecorrelated with symptoms and a humoral immune response to the NS-1protein. PCT No. WO 99/28439 by Nguyen et al. discloses the genomesequence of parvovirus B19 and fragments useful as diagnostic andimmunogenic agents. PCT No. WO 99/43362 by Barrett et al. discloses aquantitative test based on PCR amplification to demonstrate that plasmaproteins were free of human parvovirus B19 following filtration toeliminate the pathogens. PCT No. WO 01/06019 by Lazo et al. disclosesDNA oligonucleotide sequences that can hybridize to human and porcineparvovirus sequences. These oligonucleotides can serve as primers in aPCR reaction to amplify portions of the parvovirus DNA in the NS and VPregions, and as probes to detect amplified sequences. The porcineparvovirus is introduced into a sample as an internal control that isco-purified and co-amplified with the human parvovirus B19 DNA. PCT No.WO 01/14593 by Zerlauth et al. discloses a method for detectingcontaminating microorganisms, such as parvovirus B19, in pooledbiological samples by using two nucleic acid amplification processesthat have different predetermined detection sensitivities, which is usedto identify and eliminate contaminated samples. The assay first tests ascreening pool made up of combined aliquots of multiple samples by usingnucleic acid amplification to detect, at a first detection limit, thepresence of a microorganism's nucleic acid. Next, a subpool from thepositive screening pool is tested by using a second nucleic acidamplification that has a less sensitive detection limit compared to thefirst detection limit. An example of the method tested plasma by usingPCR amplification of a fragment of parvovirus B19 DNA and detectionusing fluorescently-labeled probes.

[0008] PCT No. WO 02/00924 by Tijssen et al. discloses nucleic acidsequences from various parvoviruses, including human parvovirus, thatcontain sequences coding for viral phospholipase A₂ proteins or relatedpolypeptides. These are useful for identifying agents capable ofinhibiting viral phospholipase A₂ activity or expression, includingantisense oligonucleotides, or for making improved recombinant vectorsfor gene therapy.

[0009] JP 04088985 by Sugamura et al. discloses a cloned gene encodinghuman parvovirus B19 protein VP-1, which was cloned by using PCR toamplify DNA fragments that were integrated into a cloning vector.

SUMMARY OF THE INVENTION

[0010] One aspect of the invention is a combination of at least twoseparate nucleic acid oligomers, wherein the oligomers are selected fromthe group consisting of: SEQ ID NO:4, a complementary sequence, or RNAequivalent thereof, optionally including a promoter sequence joined to a5′ terminus of the sequence, SEQ ID NO:6, a complementary sequence, orRNA equivalent thereof, optionally including a promoter sequence joinedto a 5′ terminus of the sequence, SEQ ID NO:8, a complementary sequence,or RNA equivalent thereof, optionally including a promoter sequencejoined to a 5′ terminus of the sequence, SEQ ID NO:10, a complementarysequence, or RNA equivalent thereof, optionally including a promotersequence joined to a 5′ terminus of the sequence, SEQ ID NO:12, acomplementary sequence, or RNA equivalent thereof, optionally includinga promoter sequence joined to a 5′ terminus of the sequence, SEQ IDNO:24, a complementary sequence, or RNA equivalent thereof, optionallyincluding a promoter sequence joined to a 5′ terminus of the sequence,SEQ ID NO:26, a complementary sequence, or RNA equivalent thereof,optionally including a promoter sequence joined to a 5′ terminus of thesequence, SEQ ID NO:13, a complementary sequence, or RNA equivalentthereof, SEQ ID NO:14, a complementary sequence, or RNA equivalentthereof, SEQ ID NO:15, a complementary sequence, or RNA equivalentthereof, SEQ ID NO:16, a complementary sequence, or RNA equivalentthereof, SEQ ID NO:17, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence, SEQID NO:18, a complementary sequence, or RNA equivalent thereof,optionally with a detectable label joined to the sequence, SEQ ID NO:27,a complementary sequence, or RNA equivalent thereof, optionally with adetectable label joined to the sequence, SEQ ID NO:28, a complementarysequence, or RNA equivalent thereof, optionally with a detectable labeljoined to the sequence, SEQ ID NO:30, a complementary sequence, or RNAequivalent thereof, optionally with a detectable label joined to thesequence, SEQ ID NO:31, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence, SEQID NO:32, a complementary sequence, or RNA equivalent thereof,optionally with a detectable label joined to the sequence, SEQ ID NO:34,a complementary sequence, or RNA equivalent thereof, optionally with adetectable label joined to the sequence, SEQ ID NO:36, a complementarysequence, or RNA equivalent thereof, optionally with a detectable labeljoined to the sequence, SEQ ID NO:37, a complementary sequence, or RNAequivalent thereof, optionally with a detectable label joined to thesequence, SEQ ID NO:1, a complementary sequence, or RNA equivalentthereof, and optionally a 3′ tail portion that is nonspecific for aparvovirus target sequence, SEQ ID NO:2, a complementary sequence, orRNA equivalent thereof, and optionally a 3′ tail portion that isnonspecific for a parvovirus target sequence, SEQ ID NO:20, acomplementary sequence, or RNA equivalent thereof, and optionally a 3′tail portion that is nonspecific for a parvovirus target sequence, andSEQ ID NO:21, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence. In one embodiment, the combination is selected from the groupconsisting of: SEQ ID NO:13, a complementary sequence, or RNA equivalentthereof, SEQ ID NO:24, a complementary sequence, or RNA equivalentthereof, optionally including a promoter sequence joined to the 5′terminus of the sequence, SEQ ID NO:27, a complementary sequence, or RNAequivalent thereof, optionally with a detectable label joined to thesequence, and SEQ ID NO:28, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence. Inembodiments in which the promoter sequence is joined to the 5′ terminusof an oligomer sequence, a preferred promoter sequence is SEQ ID NO:19.

[0011] Another aspect of the invention is a nucleic acid oligomerconsisting of a target-specific sequence contained in SEQ ID NO:29, acomplementary sequence, or RNA equivalent thereof, and optionally a 3′tail portion that is nonspecific for a parvovirus target sequence. Inone embodiment, the oligomer contains the target-specific sequence ofSEQ ID NO:20, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence. In another embodiment, the oligomer contains thetarget-specific sequence of SEQ ID NO:21, a complementary sequence, orRNA equivalent thereof, and optionally a 3′ tail portion that isnonspecific for a parvovirus target sequence. Yet another embodiment isan oligomer that contains the target-specific sequence of SEQ ID NO:41,a complementary sequence, or RNA equivalent thereof, and optionally a 3′tail portion that is nonspecific for a parvovirus target sequence.

[0012] One aspect of the invention is a nucleic acid oligomer consistingof a target-specific sequence of at least 25 contiguous bases containedin SEQ ID NO:1, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence.

[0013] Another aspect of the invention is a nucleic acid oligomerconsisting of a sequence contained in SEQ ID NO:33, a complementarysequence, or RNA equivalent thereof. In one embodiment, the oligomercomprises a sequence consisting of SEQ ID NO:40, a complementarysequence, or RNA equivalent thereof. Another embodiment is an oligomerthat contains the sequence of SEQ ID NO:32 or SEQ ID NO:28, acomplementary sequence, or RNA equivalent thereof. One embodiment is theoligomer that comprises a sequence consisting of SEQ ID NO:39, acomplementary sequence, or RNA equivalent thereof. In anotherembodiment, the oligomer contains the sequence of SEQ ID NO:30 or SEQ IDNO:31, a complementary sequence, or RNA equivalent thereof. In someembodiments, the oligomer has a backbone that includes at least one2′-O-methoxy linkage.

[0014] Another aspect of the invention is a nucleic acid oligomerconsisting of a sequence contained in SEQ ID NO:35, a complementarysequence, or RNA equivalent thereof. Embodiment include the oligomerthat contains the sequence of SEQ ID NO:17, SEQ ID NO:27, or SEQ IDNO:34, or a complementary sequence, or RNA equivalent thereof. Inadditional embodiments, the oligomer has a backbone that includes atleast one 2′-O-methoxy linkage.

[0015] Another aspect of the invention is a nucleic acid oligomerselected from the group consisting of SEQ ID NO:36, a complementarysequence, or RNA equivalent thereof, and SEQ ID NO:37, a complementarysequence, or RNA equivalent thereof. In some embodiments, the oligomerhas a backbone that includes at least one 2′-O-methoxy linkage.

[0016] One aspect of the invention is a method of detecting humanparvovirus B19 nucleic acid in a biological sample, comprising the stepsof providing a biological sample containing parvovirus B19 nucleic acid;amplifying in vitro a portion of the parvovirus B19 nucleic acid byusing at least one nucleic acid polymerase activity and at least onefirst amplification oligomer and one second amplification oligomer,selected from the group consisting of a first amplification oligomer ofSEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:24, or SEQ ID NO:26, each optionally including a promoter sequencejoined to a 5′ terminus of the sequence, and a second amplificationoligomer of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16;and detecting an amplified product of the parvovirus B19 nucleic acid byusing a labeled detection probe that hybridizes specifically with theamplified product, thereby indicating presence of parvovirus B19 nucleicacid in the biological sample. In some embodiments, the amplifying stepuses at least two oligomers selected from the following group ofoligomer pairs: SEQ ID NO:4 including a promoter sequence joined to the5′ terminus, with SEQ ID NO:13, SEQ ID NO:6 including a promotersequence joined to the 5′ terminus, with SEQ ID NO:13, SEQ ID NO:24including a promoter sequence joined to the 5′ terminus, with SEQ IDNO:13, and SEQ ID NO:26 including a promoter sequence joined to the 5′terminus, with SEQ ID NO:13. Embodiments include an amplifying step thatuses an amplification reaction that is substantially isothermal. In someembodiments, the detecting step uses a labeled detection probe selectedfrom the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:36, and SEQ ID NO:37. In other embodiments, the labeleddetection probe is selected from the group consisting of SEQ ID NO:17,SEQ ID NO:27, and SEQ ID NO:28. Some embodiments use a labeled detectionprobe with a backbone that includes at least one 2′-O-methoxy linkage.Embodiments of the detecting step may use a labeled detection probe thatincludes a label that is detected in a homogeneous reaction. In someembodiments, the labeled detection probe includes a chemiluminescentlabel attached to the oligomer via a linker compound. One embodiment ofthe method further includes the steps of contacting the biologicalsample with at least one capture oligomer comprising a target-specificsequence that hybridizes to a parvovirus B19 target sequence, thusforming a complex comprising the capture oligomer and parvovirus B19nucleic acid; and separating the complex from the biological samplebefore the amplifying step. In some embodiments, the method uses acapture oligomer that comprises at least 25 contiguous bases of atarget-specific sequence contained in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:20, or SEQ ID NO:21, or RNA equivalents thereof. In some embodiments,the backbone of the capture oligomer includes at least one 2′-O-methoxylinkage.

[0017] It should be understood that both the foregoing generaldescription and the following detailed description are exemplary onlyand are not restrictive of the invention. The detailed description andexamples illustrate various embodiments and explain the principles ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] This application discloses oligonucleotide sequences used asprimers for amplification and probes for detection of parvovirus B19nucleic acid sequences present in a biological sample using an assaythat includes in vitro nucleic acid amplification. A preferredembodiment of the method uses transcription-mediated nucleic acidamplification (as previously disclosed in detail in U.S. Pat. Nos.5,399,491 and 5,554,516 to Kacian et al.). Preferred methods fordetecting amplified nucleic acid include using sequence-specific probesthat hybridize specifically to a portion of the amplified sequences.Preferably, the method uses any known homogeneous detection step todetect, in a mixture, a labeled probe that is bound to an amplifiednucleic acid (e.g., as disclosed by Arnold et al., Clin. Chem.35:1588-1594 (1989); U.S. Pat. No. 5,658,737 to Nelson et al., and U.S.Pat. Nos. 5,118,801 and 5,312,728 to Lizardi et al.). This applicationalso discloses oligonucleotide sequences that are useful for capturingthe parvovirus B19 target DNA by using nucleic acid hybridizationtechniques. One embodiment of the capturing step uses magnetic particlesto separate the captured target (see U.S. Pat. No. 6,110,678 to Weisburget al.).

[0019] By “biological sample” is meant any tissue or material derivedfrom a living or dead human which may contain parvovirus nucleic acid,including, for example, sputum, peripheral blood, plasma, serum, biopsytissue including lymph nodes, respiratory tissue or exudates, or otherbody fluids, tissues or materials. The sample may be treated tophysically, chemically and/or mechanically disrupt tissue or cellstructure, thus releasing intracellular components. Sample preparationmay use a solution that contains buffers, salts, detergents and the likewhich are used to prepare the sample for analysis.

[0020] By “nucleic acid” is meant a multimeric compound comprisingnucleosides or nucleoside analogs which have nitrogenous heterocyclicbases, or base analogs, linked together by nucleic acid backbonelinkages (e.g., phosphodiester bonds) to form a polynucleotide.Conventional RNA and DNA are included in the term “nucleic acid” as areanalogs thereof. The nucleic acid backbone may include a variety oflinkages, for example, one or more of sugar-phosphodiester linkages,peptide-nucleic acid bonds (see PCT No. WO 95/32305 by Hydig-Hielsen etal.), phosphorothioate or methylphosphonate linkages or mixtures of suchlinkages in a single oligonucleotide. Sugar moieties in the nucleic acidmay be either ribose or deoxyribose, or similar compounds with knownsubstitutions, such as, for example, 2′ methoxy substitutions and 2′halide substitutions (e.g., 2′-F). Conventional nitrogenous bases (A, G,C, T, U), known base analogs (e.g., inosine; see The Biochemistry of theNucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), derivativesof purine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines and deaza- or aza-pyrimidines, pyrimidines having asubstituent at the 5 or 6 positions, purine bases having a substituentat the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; PCT No. WO93/13121 by Cook) and “abasic” residues (i.e., no nitrogenous base forone or more backbone positions) (U.S. Pat. No. 5,585,481 to Arnold etal.) are included in the term nucleic acid. That is, a nucleic acid maycomprise only conventional sugars, bases and linkages found in RNA andDNA, or may include both conventional components and substitutions(e.g., conventional bases and analogs linked via a methoxy backbone, orconventional bases and one or more base analogs linked via an RNA or DNAbackbone).

[0021] The backbone of an oligomer may affect stability of ahybridization complex (e.g., formed between of a capture oligomer to itstarget nucleic acid). Such embodiments include peptide linkages,2′-O-methoxy linkages and sugar-phosphodiester type linkages. Peptidenucleic acids are advantageous for forming a hybridization complex withRNA. An oligomer having 2′-methoxy substituted RNA groups or a 2′-fluorosubstituted RNA may have enhance hybridization complex stabilityrelative to standard DNA or RNA and is preferred for forming ahybridization complex with a complementary 2′-OH RNA. A linkage joiningtwo sugar groups may affect hybridization complex stability by affectingthe overall charge or the charge density, or by affecting stericinteractions (e.g., bulky linkages may reduce hybridization complexstability). Preferred linkages include those with neutral groups (e.g.,methylphosphonates) or charged groups (e.g., phosphorothioates) toaffect complex stability.

[0022] By “oligonucleotide” or “oligomer” is meant a nucleic acid havinggenerally less than 1,000 residues, including polymers in a size rangehaving a lower limit of about 5 nucleotide residues and an upper limitof about 500 nucleotide residues. Oligomers of some embodiments of theinvention are in a size range having a lower limit of about 5 to about15 residues and an upper limit of about 50 to 100 residues. Preferredembodiments of oligomers are in a size range having a lower limit ofabout 10 to about 25 residues and an upper limit of about 25 to about 60residues. Oligomers may be purified from naturally occurring sources,but generally are synthesized in vitro by using any well known enzymaticor chemical method. Generally, when an oligomer of the present inventionis synthesized in vitro with a 2′-O-methoxy backbone, a uracil (U) baseis used in those positions that are occupied by a thymine (T) base inthe same sequence in an oligomer synthesized with sugar-phosphodiesterlinkages, except for a 3′ T which is a standard deoxynucleotide. Thatis, methoxy oligonucleotides have a methoxy group at the 2′ position ofthe ribose moiety, and a U at the base position of a T residue in astandard DNA oligonucleotide, except when a T is present at the 3′ endof the oligomer. When an oligomer is specified as containing an “OMeT”residue, the base position is occupied by a T residue and the backbonecomprises 2′-O-methoxy linkages. Although an oligomer base sequencefrequently is referred to as a DNA sequence (i.e., contains T residues),one skilled in the art will appreciate that the corresponding RNAsequence (i.e., the same base sequence but containing U in place of T),or the complementary DNA or RNA sequences are substantially equivalentembodiments of the specified DNA sequence. Indeed, as described above,an oligomer with a 2′-O-methoxy backbone may contain a mixture of U andT bases in the same oligomer.

[0023] By “amplification oligonucleotide” or “amplification oligomer” ismeant an oligonucleotide that hybridizes to a target nucleic acid, orits complementary strand, and participates in nucleic acidamplification. Examples include primers and promoter-primers.Preferably, an amplification oligonucleotide contains at least 10contiguous bases, and more preferably at least about 12 contiguous basesbut less than about 65 bases, that hybridize specifically with a regionof the target nucleic acid sequence (or a complementary strand thereof)under standard hybridization conditions. The contiguous bases thathybridize to the target sequence are at least about 80%, preferably atleast about 90%, and more preferably about 100% complementary to thesequence to which the amplification oligonucleotide hybridizes. Anamplification oligonucleotide is preferably about 20 to about 60 nt long(e.g., 21 to 56 nt) and optionally may include modified nucleotides.

[0024] Amplification oligomers may be referred to as “primers” or“promoter-primers.” A “primer” refers to an oligonucleotide thathybridizes to a template nucleic acid and has a 3′ end that can beextended in a known polymerization reaction. The 5′ region of the primermay be non-complementary to the target nucleic acid, e.g., the 5′non-complementary region may include a promoter sequence and theoligomer is referred to as a “promoter-primer.” Those skilled in the artwill appreciate that any oligomer that can function as a primer (i.e.,an amplification oligonucleotide that hybridizes specifically to atarget sequence and has a 3′ end that can be extended by a polymerase)can be modified to include a 5′ promoter sequence, and thus function asa promoter-primer. Similarly, any promoter-primer can be modified byremoval of, or synthesis without, a promoter sequence and function as aprimer.

[0025] “Amplification” refers to any known procedure for obtainingmultiple copies of a target nucleic acid sequence or its complement orfragments thereof, and preferred embodiments amplify the targetspecifically by using sequence-specific methods. Known amplificationmethods include, for example, transcription-mediated amplification,replicase-mediated amplification, polymerase chain reaction (PCR)amplification, ligase chain reaction (LCR) amplification andstrand-displacement amplification (SDA). Replicase-mediatedamplification uses self-replicating RNA molecules, and a replicase suchas QB-replicase (e.g., see U.S. Pat. No. 4,786,600 to Kramer et al. andPCT No. WO 90/14439). PCR amplification is well known and uses DNApolymerase, sequence-specific primers and thermal cycling to synthesizemultiple copies of the two complementary strands of DNA or cDNA (e.g.,U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 to Mullis et al., andMethods in Enzymology, 1987, Vol. 155: 335-350). LCR amplification usesat least four separate oligonucleotides to amplify a target and itscomplementary strand by using multiple cycles of hybridization,ligation, and denaturation (EP Patent No. 0 320 308). SDA amplifies byusing a primer that contains a recognition site for a restrictionendonuclease which nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, followed by amplification in a series ofprimer extension and strand displacement steps (U.S. Pat. No. 5,422,252to Walker et al.) As illustrated below, preferred embodiments usetranscription-associated amplification. It will be apparent to oneskilled in the art that method steps and amplification oligonucleotidesof the present invention may be readily adapted to a variety of nucleicacid amplification procedures based on primer extension by a polymeraseactivity.

[0026] Amplification of a “fragment” or “portion” of the target sequencerefers to production of an amplified nucleic acid containing less thanthe entire target region nucleic acid sequence or its complement. Suchfragments may be produced by amplifying a portion of the targetsequence, e.g., by using an amplification oligonucleotide whichhybridizes to and initiates polymerization from an internal position inthe target sequence.

[0027] By “transcription-mediated amplification” (TMA) or“transcription-associated amplification” is meant a nucleic acidamplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. Transcription-associatedamplification generally employs RNA polymerase and DNA polymeraseactivities, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-primer, and optionally may include one ormore other amplification oligonucleotides, including “helper” oligomers.Variations of transcription-associated amplification are well known inthe art and described in detail elsewhere (see U.S. Pat. Nos. 5,399,491and 5,554,516 to Kacian et al., U.S. Pat. No. 5,437,990 to Burg et al.,U.S. Pat. No. 5,130,238 to Malek et al., U.S. Pat. Nos. 4,868,105 and5,124,246 to Urdea et al., PCT No. WO 93/22461 by Kacian et al., PCTNos. WO 88/01302 and WO 88/10315 by Gingeras et al., PCT No. WO 94/03472by McDonough et al., and PCT No. WO 95/03430 by Ryder et al.). Theprocedures of U.S. Pat. Nos. 5,399,491 and 5,554,516 are preferredamplification embodiments.

[0028] By “probe” is meant a nucleic acid oligomer that hybridizesspecifically to a target sequence in a nucleic acid, preferably in anamplified nucleic acid, under conditions that allow hybridization,thereby allowing detection of the target or amplified nucleic acid.Detection may either be direct (i.e., resulting from a probe hybridizingdirectly to the sequence) or indirect (i.e., resulting from a probehybridizing to an intermediate molecular structure that links the probeto the target). The probe's “target” generally refers to a sequencewithin or a subset of an amplified nucleic acid sequence whichhybridizes specifically to at least a portion of a probe oligomer bystandard hydrogen bonding (i.e., base pairing). A probe may comprisetarget-specific sequences and other sequences that contribute tothree-dimensional conformation of the probe (e.g., U.S. Pat. Nos.5,118,801 and 5,312,728 to Lizardi et al., and U.S. Pat. No. 6,361,945B1 to Becker et al.). Sequences are “sufficiently complementary” if theyallow stable hybridization in appropriate hybridization conditions of aprobe oligomer to a target sequence that is not completely complementaryto the probe's target-specific sequence.

[0029] By “sufficiently complementary” is meant a contiguous nucleicacid base sequence that is capable of hybridizing to another basesequence by hydrogen bonding between a series of complementary bases.Complementary base sequences may be complementary at each position inthe oligomer sequence by using standard base pairing (e.g., G:C, A:T orA:U) or may contain one or more residues that are not complementary(including abasic positions), but in which the entire complementary basesequence is capable of specifically hybridizing with another basesequence in appropriate hybridization conditions. Contiguous bases arepreferably at least about 80%, more preferably at least about 90%, andmost preferably 100% complementary to a sequence to which an oligomer isintended to hybridize. Those skilled in the art can readily chooseappropriate hybridization conditions which can be predicted based onbase sequence composition, or be determined by using routine testing(e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual,2^(nd) ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57,particularly §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0030] By “capture oligonucleotide” or “capture oligomer” or “captureprobe” is meant a nucleic acid oligomer that hybridizes specifically toa target nucleic acid to be captured and provides a means for isolatingand/or concentrating the target from other sample components.Embodiments of capture oligomers include two binding regions: atarget-binding region and an immobilized probe-binding region, wherebythe capture oligomer forms a hybridization complex in which thetarget-binding region of the capture oligomer binds to the targetsequence and the immobilized probe-binding region binds to an oligomerimmobilized on a solid support (see U.S. Pat. Nos. 6,110,678 and6,280,952 to Weisburg et al.). Although the target-binding region andimmobilized probe-binding region are usually on the same captureoligomer, the two functional regions may be present on two differentoligomers joined together by one or more linkers. For example, animmobilized probe-binding region may be present on a first oligomer, atarget-binding region may be present on a second oligomer, and the twooligomers are joined by hydrogen bonding with a third oligomer that is alinker that hybridizes specifically to sequences of the first and secondoligomers. The target-binding region of a capture probe may also bereferred to as a target-specific portion of the capture probe and theimmobilized probe-binding region may be referred to as a tail portion.Embodiments of tail portions include homopolymers (e.g., poly-dT orpoly-dA) or non-homopolymers (e.g., T₁₋₃A₃₀), preferably attached to the3′ end of the target-specific portion of the oligomer.

[0031] By “immobilized probe” or “immobilized oligomer” is meant anucleic acid oligomer that joins, directly or indirectly, a captureoligomer to an immobilized support. An immobilized probe joined to asolid support facilitates separation of bound target sequence fromunbound material in a sample. Any known solid support may be used, suchas matrices and particles in solution, e.g., nitrocellulose, nylon,glass, polyacrylate, mixed polymers, polystyrene, silane polypropyleneand metal particles, preferably, magnetically attractable particles.Preferred supports are monodisperse paramagnetic spheres (e.g., uniformsize ±5%), to provide consistent results, to which an immobilized probeis joined directly (e.g., via a direct covalent linkage, chelation, orionic interaction), or indirectly (e.g., via one or more linkers), wherethe linkage or interaction is stable during nucleic acid hybridizationconditions.

[0032] By “separating” or “purifying” is meant that one or morecomponents of the biological sample are removed from at least one othercomponent of the sample. Sample components generally include an aqueoussolution of nucleic acids, salts, proteins, carbohydrates, and lipids. Astep of separating or purifying a nucleic acid removes at least about70%, preferably at least about 90% and, more preferably, at least about95% of the other components in the sample.

[0033] By “label” is meant a molecular moiety or compound that can bedetected or can lead to a detectable signal. A label is joined, directlyor indirectly, to a nucleic acid probe. Direct labeling uses bonds orinteractions that link the label to the probe, including covalent bondsor non-covalent interactions, such as hydrogen bonds, hydrophobic andionic interactions, or through formation of chelates or coordinationcomplexes. Indirect labeling uses a bridging moiety or “linker” (e.g.,oligonucleotide or antibody), to link the label and probe. Linkers canbe used to amplify a detectable signal. Labels are any known detectablemoiety, e.g., radionuclide, ligand (e.g., biotin, avidin), enzyme orenzyme substrate, reactive group, or chromophore, such as a dye ordetectable particle (e.g., latex beads or metal particles), luminescentcomponds (e.g., bioluminescent, phosphorescent or chemiluminescentlabels) and fluorescent compounds. Preferably, the label on a labeledprobe is detectable in a homogeneous reaction (i.e., in a mixture, boundlabeled probe exhibits a detectable change, such as stability ordifferential degradation, compared to unbound labeled probe). Oneembodiment of a label for use in a homogenous assay is achemiluminescent compound (e.g., described in detail in U.S. Pat. No.5,656,207 to Woodhead et al., U.S. Pat. No. 5,658,737 to Nelson et al.,and U.S. Pat. No. 5,639,604 to Arnold, Jr., et al.). Preferredchemiluminescent labels are acridinium ester (AE) compounds, such asstandard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE, 1- or3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE,ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE). Synthesis and methods of attaching labels to nucleic acidsand detecting labels are well known in the art (e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Habor, N.Y., 1989), Chapter 10; U.S. Pat.No. 4,581,333 to Kourilsky et al., U.S. Pat. No. 5,658,737 to Nelson etal., U.S. Pat. No. 5,656,207 to Woodhead et al., U.S. Pat. No. 5,547,842to Hogan et al., U.S. Pat. No. 5,283,174 to Arnold, Jr. et al., and EPPatent Pub. No. 0747706 by Becker et al.). Another embodiment of a labelfor use in a homogenous assay is a fluorescent compound attached to aprobe with a quencher compound in functional proximity to thefluorescent label when the probe is not hybridized to its target (e.g.,U.S. Pat. Nos. 5,118,801 and 5,312,728 to Lizardi et al., and U.S. Pat.No. 6,361,945 B1 to Becker et al.).

[0034] A “homogeneous detectable label” refers to a label whose presencecan be detected in a homogeneous fashion based upon whether the labeledprobe is hybridized to a target sequence (i.e., can be detected withoutphysically removing unhybridized label or labeled probe). Embodiments ofhomogeneous detectable labels and methods of detecting them have beendescribed (U.S. Pat. No. 5,283,174 to Arnold et al., U.S. Pat. No.5,656,207 to Woodhead et al., U.S. Pat. No. 5,658,737 to Nelson et al.,U.S. Pat. Nos. 5,118,801 and 5,312,728 to Lizardi et al., and U.S. Pat.No. 6,361,945B1 to Becker et al.).

[0035] By “consisting essentially of” is meant that additionalcomponent(s) and method step(s)] that do not materially change the basicand novel characteristics of the present invention may be included. Suchcharacteristics include salts, buffering agents, nucleic acid oligomersand similar biochemical reagents that do not have a material effect onthe characteristics of the claimed components or method steps describedherein that detect parvovirus B19 nucleic acid sequences, includingnucleic amplification products derived from parvovirus B19 DNA, with asensitivity of about 100 to 500 copies of parvovirus B19 DNA in thestarting material. Similarly, additional method steps that do not have amaterial effect on the basic nature of the assay may be included.

[0036] Assays of the present invention detect human parvovirus presentin a biological sample (e.g., blood, serum, plasma, sputum, bronchiallavage). In one embodiment, the assay detected parvovirus B19 DNA inplasma samples, either pooled or from individual donors. To prepareplasma specimens, whole blood samples were centrifuged using standardmethods, and the plasma was stored at 4° C. or −20° C. before testing.To lyse viral particles in the specimen, a lysing reagent containing adetergent was mixed with the specimen to release the parvovirus B19 DNAfrom viral particles. Specimen processing may combine viral lysis withpurification of the viral target DNA by including a capture oligomer andimmobilized oligomer in the lysing reagent. Then the method includes atarget capture step in which the parvovirus B19 DNA is hybridizedspecifically to the capture oligomer, which is then hybridized to theimmobilized oligomer, and the bound complex (i.e., immobilized oligomer,capture oligomer, and viral target DNA) is substantially separated fromother sample components. Residual sample components are washed away bywashing the solid support with the bound parvovirus-containing complex.Thus, the viral target DNA is separated form other sample components andconcentrated in the bound complexes, without releasing the boundparvovirus B19 nucleic acid from the solid support.

[0037] Typical sample processing involved the following steps (describedin detail in U.S. Pat. No. 6,110,678). Viral particles in body fluid(e.g., 0.5 ml of plasma) were lysed upon contact at 60° C. with targetcapture reagent (790 mM HEPES, 680 mM LiOH, 10% lithium lauryl sulfate(LLS), 230 mM succinate, at least one capture probe at 7 pm/ml, and 100μg/ml of poly-dT₁₄ bound to magnetic particles (SERADYN™, Indianapolis,Ind.)). Capture oligomers were composed of 5′ target-specific sequence(e.g., SEQ ID NOs. 1, 2, 20 and 21) that is complementary to andhybridizes specifically to a target sequence that is a portion of theparvovirus B19 genome sequence, and a 3′ tail sequence (e.g., oligo-dA)that hybridizes to the complementary oligmer (e.g., oligo-dT) attachedto the solid support. Other preferred embodiments of capture probes areoligomers that contain a target-complementary sequence of 27 to 33nucleotides that includes SEQ ID NO:41, and oligomers that bindspecifically to the target sequences shown in SEQ ID NO:22 and SEQ IDNO:29. Target capture hybridization occurs in this reaction mixture byincubating the mixture at a first temperature (60° C.), allowing thecapture oligomer to bind specifically to its complementary targetsequence in parvovirus B19 DNA. Then, the mixture was cooled to 40° C.or lower (e.g., room temperature) to allow the 3′ tail of the captureoligomer to hybridize to its complementary oligomer on the particle.Following the second hybridization, the mixture is treated to separatethe solid support with its bound complex of nucleic acids from the othersample components, e.g., by using gravitational, centrifugal, ormagnetic separation. Generally, separation employed a rack that containsa magnet to pull the magnetic particles with bound nucleic acidcomplexes to the side of the tube. Then the supernatant was removed andthe bound complexes on the particles were washed with 1 ml of a washingbuffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) absoluteethanol, 0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, 150 mMNaCl, 0.1% sodium dodecyl sulfate (SDS), pH 7.5) by suspending themagnetic particles in washing buffer, separating particles to the tubeside, and removing the supernatant.

[0038] Following sample preparation, amplification of the parvovirus B19DNA target was achieved by using a pair of amplification oligomers thatdefine the 5′ and 3′ ends of the region amplified by in vitroenzyme-mediated nucleic acid synthesis. One embodiment uses atranscription-mediated amplification (TMA) method, substantially asdescribed in U.S. Pat. Nos. 5,399,491 and 5,554,516, which is asubstantially isothermal system that produces a large number ofamplification products (RNA transcripts) that can be detected.Embodiments of the method used mixtures of amplification oligomers inwhich at least one promoter primer is selected from the group consistingof SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQID NO:23, and SEQ ID NO:25, which is combined with at least one primerselected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16. Other embodiments used mixtures ofamplification oligomers in which at least one promoter primer isselected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQID NO:9, which is combined with at least one primer selected from thegroup consisting of SEQ ID NO:13 and SEQ ID NO:16. Some embodiments usedmixtures of amplification oligomers in which at least one promoterprimer is selected from the group consisting of SEQ ID NO:3, SEQ IDNO:23, and SEQ ID NO:25, which is combined with a primer consisting ofSEQ ID NO:13. One skilled in the art will appreciate that oligomersconsisting of the target-specific portions of promoter primers canhybridize to the target sequence and provide a 3′ end to function as aprimer for enzyme-mediated nucleic acid amplification, independent ofits promoter sequence (SEQ ID NO:19). Such oligomers include thesequences of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:24, and SEQ ID NO:26.

[0039] Amplifying the target nucleic acid by transcription-mediatedamplification produces many strands of nucleic acid from a single copyof target nucleic acid, thus permitting detection of the target bydetecting probes that hybridize to the amplified sequences. Generally,the reaction mixture includes the target nucleic acid and two primers,including at least one promoter primer, reverse transcriptase and RNApolymerase activities, nucleic acid synthesis substrates(deoxyribonucleoside triphosphates and ribonucleoside triphosphates) andappropriate salts and buffers in solution to produce multiple RNAtranscripts from a nucleic acid template. Briefly, a firstpromoter-primer hybridizes specifically to a portion of the targetsequence and reverse transcriptase that includes RNase H activitycreates a first strand cDNA by 3′ extension of the promoter-primer. ThecDNA is hybridized with a second primer downstream from the firstpromoter primer and a new DNA strand is synthesized from the 3′ end ofthe second primer using the reverse transcriptase to create a dsDNAhaving a functional promoter sequence at one end. RNA polymerase bindsto the dsDNA at the promoter sequence and transcribes multipletranscripts or “amplicons.” These amplicons are further used in theamplification process, serving as a template for a new round ofreplication, to ultimately generate large amounts of single-strandedamplified nucleic acid from the initial target sequence (e.g., 100 to3,000 copies of RNA synthesized from a single template). The processuses substantially constant reaction conditions (i.e., substantiallyisothermal). A typical 100 μl amplification reaction uses 75 μl of anamplification reagent mixture (11.6 mM Tris Base, 15.0 mM Tris-HCl, 22.7mM MgCl₂, 23.3 mM KCl, 3.33% glycerol, 0.05 mM Zn-acetate (dihydrate),0.665 mM each of dATP, dCTP, dGTP, and dTTP, 5.32 mM each of ATP, CTP,GTP, and UTP, pH 7) and 25 μl of an enzyme reagent mixture (700 U of T7RNA polymerase, 1400 U of reverse transcriptase from Moloney MurineLeukemia Virus (MMLV-RT), 16 mM HEPES (free acid, dihydrate), 70 mMN-acety-L-cysteine, 3 mM EDTA, 0.05% (w/v) Na-azide, 20 mM Tris base, 50mM KCl, 20% (v/v) anhydrous glycerol, 10% (v/v) TRITON®) X-102, and 150mM trehalose (dihydrate), pH 7), preferably mixed with the capturedtarget nucleic acid retained on the solid particles. For the enzymaticactivities, 1 U of T7 RNA polymerase incorporates 1 nmol of ATP into RNAin 1 hr at 37° C. using a DNA template containing a T7 promoter, and 1 Uof MMLV-RT incorporates 1 nmol of dTTP into DNA in 10 min at 37° using200-400 μmol oligo dT-primed poly(A) as a template.

[0040] Following amplification, the amplified sequences generated fromthe parvovirus B19 target DNA are detected, preferably by hybridizationwith at least one labeled nucleic acid probe that hybridizesspecifically to a portion of the amplified sequence. Probe embodimentsinclude those having a T_(m) in the range of about 80° C. to 85° C. Someprobe embodiments include oligomers having sequences of SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:37. Otherembodiments are oligomers of 20 to 22 nucleotides that include sequencescontained in SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40. Other probeembodiments include oligomers of at least 21 contiguous nucleotidescontained in SEQ ID NO:33, and oligomers of 20 to 25 contiguousnucleotides contained in SEQ ID NO:35. Detection of the labeled probe ispreferably accomplished by detecting a label that can be detected in ahomogeneous reaction. Therefore, some embodiments include probes labeledwith an acridinium ester (AE) compound using well-known methods thatallow homogeneous detection (e.g., labels and detection methods aredescribed in detail in U.S. Pat. No. 5,283,174 to Arnold, Jr., et al.,U.S. Pat. No. 5,656,207 to Woodhead et al., and U.S. Pat. No. 5,658,737to Nelson et al.). A chemiluminescent AE compound is attached to theprobe sequence via a linker compound (substantially as described in U.S.Pat. Nos. 5,585,481 and 5,639,604 to Arnold, Jr., et al., e.g., seecolumn 10, line 6 to column 11, line 3, and Example 8). In someembodiments, AE compound labels were linked to SEQ ID NO:17 betweenpositions 7 and 8 or positions 11 and 12, and to SEQ ID NO:18 betweenpositions 11 and 12. Additional embodiments of labeled probes aredescribed in Example 9. In one embodiment, the labeled probe oligomerhas at least one 2′-O-methoxy linkage in the nucleic acid backbone(e.g., SEQ ID NO:18 synthesized with a 2′-O-methoxy backbone). In atypical detection step, the probe reagent included 100 mM succinate, 2%(w/v) LLS, 230 mM LiOH (monohydrate), 15 mM 2,2′-dithiodipyridine(ALDRITHIOL-2),1.2 M LiCl, 20 mM EDTA, 20 mM EGTA, 3% (v/v) absoluteethanol, brought to about pH 4.7 with LiOH, and the selection reagentused for hydrolyzing the label on unbound probe included 600 mM boricacid, 182 mM NaOH, 1% (v/v) TRITON® X-100. The signal was detected asrelative light units (RLU) using a luminometer (e.g., LEADER™ 450HC+,Gen-Probe Incorporated, San Diego, Calif.).

[0041] To select DNA sequences appropriate for use as capture oligomers,amplification oligomers and detection probes, known parvovirus B19 DNAsequences, including partial or complementary sequences, available frompublicly accessible databases (e.g., GenBank) were aligned by matchingregions of the same or similar sequences and compared using well knownmolecular biology techniques. Although sequence comparisons may befacilitated by use of algorithms, those skilled in the art can readilyperform such comparisons manually and visually. Generally, portions ofsequences that contain relatively few variants between the comparedsequences were chosen as a basis for designing synthetic oligomers foruse in the present invention. Other considerations in designingoligomers included the relative GC content (which affects T_(m)) and therelative absence of predicted secondary structure (which potentiallyform intramolecular hybrids) within a sequence, as determined by usingwell known methods.

[0042] In one embodiment, the assay is carried out in a single tubeusing a 0.5 to 1 ml sample of body fluid (e.g., plasma) to detect targetparvovirus B19 DNA at a sensitivity of about 100 to 500 copies/ml oftarget DNA per reaction. In other embodiments, the assay detected highernumbers of target parvovirus B19 DNA in the sample, which may be apooled sample of individual samples.

[0043] Unless defined otherwise, all scientific and technical terms usedherein have the same meaning as commonly understood by those skilled inthe relevant art. General definitions of many of the terms used hereinare provided in Dictionary of Microbiology and Molecular Biology, 2nded. (Singleton et al., 1994, John Wiley & Sons, New York, N.Y.), TheHarper Collins Dictionary of Biology (Hale & Marham, 1991, HarperPerennial, New York, N.Y.), and Taber's Cyclopedic Medical Dictionary,17th ed. (F. A. Davis Co., Philadelphia, Pa., 1993). Unless mentionedotherwise, the techniques employed or contemplated herein are standardmethodologies well known to one of ordinary skill in the art. Thefollowing examples illustrate some of the preferred embodiments of theinvention and are provided for illustration only.

EXAMPLE 1 Target Capture of Parvovirus B19 DNA

[0044] Capture probes were synthesized having sequences of SEQ ID NO: 1and SEQ ID NO:2, both having 3′ dA₃₀ tails, using standard in vitro DNAsynthesis reactions. Using the target capture methods described above,the capture oligomers were mixed with human plasma samples obtained fromuninfected donors, each sample spiked with a known number of copies oflive parvovirus B19 (2,000,1,000, 500 or 0 in the negative control). Thevirions were lysed by mixing the plasma sample (generally 0.5 ml) withan equal volume of target capture reagent containing each of the captureprobes separately (3.5 pm per reaction). Following capture byhybridization at about 60° C. for about 20 min and then at 18-25° C. forabout 10-20 min, the magnetic particles with attached hybridizationcomplexes were washed twice as described above. The parvovirus targetsequence in the complexes retained on the particles was amplified in aTMA reaction performed substantially as described in Example 2, and theamplified target sequences were detected by hybridization with aAE-labeled probe (SEQ ID NO:17). The results (RLU detected from bounddetection probe) are shown in Table 1. These results (RLU of about1.5×10⁶) show that both capture probes specifically bind to andeffectively capture parvovirus B19 DNA from a sample compared to thenegative control (RLU about 2×10⁴). TABLE 1 Detection of labeled probe(RLU) bound to parvovirus B19 DNA following target capture. B19 copiesA₃₀ Capture Probe SEQ A₃₀ Capture Probe SEQ per reaction NO:1 NO:2 2,0001.56 × 10⁶ 1.61 × 10⁶ 1,000 1.42 × 10⁶ 1.53 × 10⁶ 500 1.58 × 10⁶ 1.59 ×10⁶ 0 1.98 × 10⁴ 2.44 × 10⁴

EXAMPLE 2 Amplification of Parvovirus B19 DNA and Detection of Amplicons

[0045] A known amount of parvovirus B19 target nucleic acid (denaturedssDNA at 1000, 500, 250, 100, 50 and 0 copies per reaction tube) wasamplified in a TMA reaction using reagents (75 μl per reaction) asdescribed above containing a promoter primer of SEQ ID NO:3 and a primerof SEQ ID NO:13 (7.5 pmol each), and covered with 200 μl of inertsilicone oil to prevent evaporation. The mixture was incubated 10 min at60° C., then 10 min at 42° C. and then 25 μl of enzyme reagent was addedand the tubes were mixed by hand and then incubated 60 min at 42° C.Following amplification the samples were incubated at 60° C. and 100 μlof probe reagent was added containing probe of SEQ ID NO:17. The mixturewas incubated 20 min at 60° C. and then 300 μl of selection reagent wasadded, mixed, and incubated 10 min at 60° C. and 10 min at roomtemperature before detecting the signal (RLU) as described above.

[0046] Following such amplification and detection steps, the averagesignal detected from five replicate samples for each condition were:1,000 copies produced 6.78×10⁶ RLU, 500 copies produced 5.56×10⁶ RLU,250 copies produced 4.42×10⁶ RLU, 100 copies produced 1.06×10⁶ RLU, and50 copies produced 2.03×10⁴ RLU, all compared to a negative control (0copies) that produced 1.64×10⁴ RLU. These results show that thesensitivity of the amplification and detection assay is at least 100copies of target.

EXAMPLE 3 Detection of Parvovirus B19 in an Amplification Assay thatUses Target Capture

[0047] In this example, the target capture assay performed substantiallyas described in Example 1 was combined with amplification and detectionsteps performed substantially as described in Example 2. The copies ofvirion were known (500, 250,100 and 0 for the negative control) for eachsample tested. The capture probes were as in Example 1, and thecombinations of amplification oligomers were SEQ ID NO:3 with SEQ IDNO:13, and SEQ ID NO:5 with SEQ ID NO:13. The detection probe was SEQ IDNO:17. The results shown in Table 2 are for an average of 5 samples foreach of the conditions tested. These results show that the sensitivityof the assay is about 250 copies of target parvovirus DNA in the sampleor better (i.e., capable of detecting 100 copies per sample). The primerset of SEQ ID NO:3 plus SEQ ID NO:13 had sensitivity of better than 250copies of virus in capture from plasma, as well has stable, reproduciblesignal with 100 copies sensitivity in an amplification and detectionassay as described in Example 2. TABLE 2 Detected signal (RLU) fortarget capture plus amplification assays. Oligomers Copies of ParvovirusB19 Capture Amplification 500 250 100 0 SEQ ID NO:1 SEQ ID NO:3 1.30 ×10⁶ 1.30 × 10⁶ 9.86 × 10⁵ 3.30 × 10⁴ SEQ ID NO:13 SEQ ID NO:5 2.37 × 10⁶2.91 × 10⁵ 2.52 × 10⁵ 4.66 × 10⁴ SEQ ID NO:13 SEQ ID NO:2 SEQ ID NO:39.65 × 10⁵ 8.88 × 10⁵ 7.85 × 10⁵ 1.15 × 10⁵ SEQ ID NO:13 SEQ ID NO:52.06 × 10⁵ 1.03 × 10⁵ 4.14 × 10⁴ 3.51 × 10³ SEQ ID NO:13

EXAMPLE 4 Clinical Specimen Testing

[0048] To determine assay specificity and prevalence of parvovirus B19DNA in a normal blood donor population, the TMA-based assay wasperformed substantially as described in Example 3 (using amplificationoligomers of SEQ ID Nos. 3 and 13) on clinical and commerciallyavailable plasma samples. The assay was performed on 468 random blooddonor specimens that were negative for the presence of HIV and HCV(obtained from the Community Blood Center of Greater Kansas City, KansasCity, Mo.). Three specimens of 468 (0.6%) were positive for parvovirusB19 (i.e., provided a signal equivalent to that detected for at least100 copies of target when compared to positive controls). All threepositive samples provided positive results when they were retested usingthe same TMA-based assay and when tested using a PCR-based assay thatamplifies a different target sequence of parvovirus B19.

[0049] The sensitivity of this TMA-based assay was estimated based on astandardized parvovirus B19 DNA specimen (obtained from the American RedCross and quantified by the National Genetics Institute). Detection ofpositive samples containing 500 copies/ml and 200 copies/ml of theparvovirus B19 standard in the assay was 100% (10 of 10 tests) and 80%(8 of 10 tests), respectively. Another standard (obtained from theNational Institute for Biological Standards and Controls, or NIBSC) thatcontains 1,000 genome equivalents/ml also tested positive in this assay.These results show that the assay has the sensitivity and specificityrequired for detecting human parvovirus B19 in a blood screeningenvironment.

[0050] Several specimens that were antibody-positive for parvovirus B19were also tested using this TMA-based assay. Four plasma samplespositive for anti-parvovirus IgM (LM 00PP751, 05256-520, 05254-520, and05259-520, from SeraCare Life Sciences, Inc., Oceanside, Calif.) allgave positive results for parvovirus B19 DNA when tested using theTMA-based assay, compared to the NIBSC standard as a positive control.Six specimens (obtained from Chiron Corp., Emeryville, Calif.) positivefor anti-parvovirus antibodies and parvovirus B19 DNA (based on aPCR-based test) were diluted 1 to 10 into negative human plasma. Thediluted samples were assayed using the TMA-based assay, and all sixspecimens tested positive for parvovirus B19, showing the high level ofsensitivity of the assay. Six plasma specimens (from BioClinicalPartners, Inc., Franklin, Mass., USA) positive for anti-parvovirus B19IgM were also tested using the TMA-based assay. All six plasmas testednegative for parvovirus B19 nucleic acid, showing that the virus hadcleared from the donors even though their plasma retained antibodies toparvovirus B19.

EXAMPLE 5 Sample Preparation Using Various Target Capture Oligomers

[0051] This example shows that various oligomers can be used alone or incombination in an initial step of the assay, i.e., capture of parvovirusB19 DNA from a sample by using hybridization to a capture oligomer. Inthese experiments, oligomers of SEQ ID NO:1, SEQ ID NO:20 and SEQ IDNO:21 were synthesized with a 3′ poly(A) tail portion and used tocapture parvovirus B19 DNA from a sample using procedures substantiallyas described previously (U.S. Pat. No. 6,110,678). Briefly, a plasmasample containing parvovirus B19 was mixed with a lysing and capturereagent containing one or more of the capture oligomers and the mixturewas incubated (60° C., 20 min) to allow the capture oligomers tohybridize to the parvovirus target DNA. The mixture also containedhomopolymeric oligomers complementary to the 3′-tail portion of thecapture oligomer and attached to magnetic particles. These homopolymericcomplementary sequences hybridized in a second hybridization reaction(25° C., 14-20 min) and the hybridization complexes attached to themagnetic particles were separated from the rest of the sample and washed(e.g., twice with 1 ml of a buffer than maintains the hybridizationcomplexes on the particles) before proceeding to amplification. In theseexperiments, 3.5 pmol per reaction of each of the capture oligomerstested as follows: reactions included each of SEQ ID Nos 1, 20 and 21individually, and in the various possible combinations (SEQ ID Nos 1 and20, SEQ ID Nos 1 and 21, SEQ ID Nos 20 and 21, and SEQ ID Nos 1, 20 and21). After the samples were treated with the capture reagent, themagnetic particles with the attached hybridization complexes wereincubated in an amplification mixture containing 15 pmol per reaction ofeach of a promoter primer of SEQ ID NO:23 and a primer of SEQ ID NO:13,and the appropriate salts, nucleotides and enzymes for a one-hour TMAreaction (substantially as described in detail previously in U.S. Pat.Nos. 5,399,491 and 5,554,516). The detection probe of SEQ ID NO:17labeled with 2-methyl-AE between nt 7 and nt 8 was added (0.1 pmol perreaction) and incubated (60° C., 20 min) with the amplification productsto allow hybridization, and the chemiluminescent signal was detected(RLU) as described in detail previously (U.S. Pat. Nos. 5,283,174,5,656,207, and 5,658,737).

[0052] In a first set of experiments, the tested human plasma samplescontained no parvovirus B19 (negative samples) or 1,000 copies perreaction of parvovirus B19 (positive samples), which were prepared bydilution from a stock sample of infected plasma (from the American RedCross) which had been titrated by comparison with a standardized sample(from NIBSC). Ten replicate samples were tested for each of theconditions. The detected results (RLU mean±standard deviation) are shownin the table that follows. TABLE 3 Results of Assays Performed UsingDifferent Capture Oligomers. Capture Oligomers Negative Samples PositiveSamples SEQ ID NO:1 2,461 ± 1,252 4,067,173 ± 163,492 SEQ ID NO:20 2,137± 360   3,893,205 ± 477,513 SEQ ID NO:21 4,774 ± 6,970 3,954,41 6 ±468,324  SEQ ID NO:1 + 5,285 ± 4,911 4,078,141 ± 269,686 SEQ ID NO:20SEQ ID NO:1 + 2,560 ± 1,002 4,093,581 ± 271,294 SEQ ID NO:21 SEQ IDNO:20 + 2,164 ± 301   4,000,996 ± 361,454 SEQ ID NO:21 SEQ ID NO:1 +4,291 ± 4,919 3,994,533 ± 116,348 SEQ ID NO:20 + SEQ ID NO:21

[0053] In a second set of experiments, the plasma samples contained noparvovirus B19 (negative samples) or varying amounts of parvovirus B19(1,000, 500, 250,100 and 50 copies per reaction), prepared by dilutionfrom the stock sample described above. For each of the conditions, fivereplicate samples were tested for those containing 1,000 and 0 copies ofparvovirus B19, and ten replicate samples were tested for all theothers. The detected results (RLU mean±standard deviation) are shownbelow. For all of the negative controls (0 copies per reaction), thedetected background was in the range of 2,277±215 to 4,724±3,889 RLU.TABLE 4 Sensitivity of Assays Performed Using Different CaptureOligomers Capture Oligomers Copies of Parvovirus B19 Per Reaction SEQ IDNOs 1,000 500 250 100 50 1 4.04 × 10⁶ 3.80 × 10⁶ 3.55 × 10⁶ 1.89 × 10⁶8.04 × 10⁵ ± 4.18 × 10⁵   ± 5.01 × 10⁵   ± 8.04 × 10⁵   ± 1.55 × 10⁶   ±1.34 × 10⁶   20 4.00 × 10⁶ 3.89 × 10⁶ 2.78 × 10⁶ 2.06 × 10⁶ 1.85 × 10⁶ ±3.60 × 10⁵   ± 5.63 × 10⁵   ± 1.22 × 10⁵   ± 1.59 × 10⁶   ± 1.73 × 10⁵  21 4.27 × 10⁶ 4.00 × 10⁶ 3.25 × 10⁶ 1.26 × 10⁶ 7.13 × 10⁵ ± 5.85 × 10⁴  ± 3.22 × 10⁵   ± 1.24 × 10⁶   ± 1.05 × 10⁶   ± 1.28 × 10⁶   1 and 204.14 × 10⁶ 3.51 × 10⁶ 3.57 × 10⁶ 2.10 × 10⁶ 1.13 × 10⁶ ± 1.71 × 10⁶   ±1.34 × 10⁵   ± 9.30 × 10⁵   ± 1.66 × 10⁶   ± 1.52 × 10⁶   1 and 21 4.28× 10⁶ 3.78 × 10⁶ 3.23 × 10⁶ 1.60 × 10⁶ 1.44 × 10⁶ ± 8.45 × 10⁴   ± 1.16× 10⁶   ± 1.08 × 10⁶   ± 1.33 × 10⁶   ± 1.60 × 10⁶   20 and 21 4.15 ×10⁶ 4.26 × 10⁶ 2.68 × 10⁶ 1.55 × 10⁶ 1.06 × 10⁶ ± 2.10 × 10⁵   ± 1.49 ×10⁵   ± 1.76 × 10⁶   ± 1.69 × 10⁶   ± 1.28 × 10⁶   1, 20 and 21 4.24 ×10⁶ 4.35 × 10⁶ 2.56 × 10⁶ 2,529,303 9.62 × 10⁵ ± 1.30 × 10⁵   ± 1.09 ×10⁵   ± 1.59 × 10⁶   ± 1,652,537 ± 1.46 × 10⁶  

[0054] The results of these experiments show that when the assay wasperformed with any of the three capture oligomers, alone or in amixture, each format detected the presence of parvovirus B19. The assaysresulted in positive signals for all samples that contained 250 to 1,000copies/ml, for 80 to 90% of samples that contained 100 copies/ml, andfor 50 to 70% of samples that contained 50 copies/ml.

EXAMPLE 6 Amplification of Parvovirus B19 Sequences Using DifferentAmplification Oligomers

[0055] This example shows that different combinations of amplificationoligomers serving as primers can amplify efficiently the targetsequences in parvovirus B19 DNA. The target sequences were amplified byusing a combination of primers that had the parvovirus-specific portionsof SEQ ID NO:24 (AGTACCGGGTAGTTGTACGCTAACT) or SEQ ID NO:26(CTAGGTTCTGCATGACTGCTACTGGA) and SEQ ID NO:13 (CCCCTAGAAAACCCATCCTCT).

[0056] Samples were prepared by mixing a human plasma that does notcontain parvovirus (negative control) with aliquots of parvovirus B19 toproduce samples containing 10,000, 5,000, 1,000, 500, 250, 100, 50, and25 copies of parvovirus B19 per ml. As a positive control, standardsamples containing 1,000 copies of parvovirus B19 per ml were alsoassayed. The samples were mixed with an oligomer (SEQ ID NO:1) which wasallowed to hybridize to the parvovirus B19 DNA, and then thehybridization complex containing the parvovirus B19 DNA was separatedfrom the sample by hybridizing it to an oligomer attached to a magneticbead, substantially as described previously (U.S. Pat. No. 6,110,678).

[0057] The amplification assays were performed using the TMA systemsubstantially as described above using 15 pmol each of the promoterprimer of SEQ ID NO:23 and SEQ ID NO:13, or the promoter primer of SEQID NO:25 and SEQ ID NO:13. Following the one-hour amplificationreaction, the mixtures were hybridized with a detection probe of SEQ IDNO:17 labeled with a chemiluminescent compound between nt 7 and 8 (using5.5×10⁹ RLU per reaction), and the relative light unit (RLU) signalswere detected as described above. For the positive and negativecontrols, 5 replicate samples were tested. For the experimental samples,10 replicates were tested for each condition. The results of theseassays (RLU mean±standard deviation) are shown in the table thatfollows. TABLE 5 Assay Results Obtained Using Different AmplificationOligomers Parvovirus B19 SEQ ID NO:13 and SEQ ID NO:13 and copies/ml SEQID NO:23 Primers SEQ ID NO:25 Primers 1,000 (positive 3.87 × 10⁶ ± 1.89× 10⁵ 2.53 × 10⁶ ± 7.94 × 10⁵ control) 10,000 4.20 × 10⁶ ± 8.27 × 10⁴4.03 × 10⁶ ± 7.29 × 10⁵ 5,000 4.07 × 10⁶ ± 2.73 × 10⁵ 3.97 × 10⁶ ± 1.15× 10⁵ 1,000 3.80 × 10⁶ ± 7.38 × 10⁵ 2.77 × 10⁶ ± 6.63 × 10⁵ 500 3.17 ×10⁶ ± 1.09 × 10⁶ 1.82 × 10⁶ ± 1.13 × 10⁶ 250 2.90 × 10⁶ ± 7.57 × 10⁵1.07 × 10⁶ ± 5.83 × 10⁵ 100 1.73 × 10⁶ ± 1.54 × 10⁶ 3.79 × 10⁵ ± 4.73 ×10⁵ 50 1.74 × 10⁶ ± 1.74 × 10⁴ 2.34 × 10⁵ ± 5.96 × 10⁵ 25 2.20 × 10⁵ ±5.66 × 10⁵ 2.52 × 10⁵ ± 7.83 × 10⁵ 0 (negative 2.73 × 10³ ± 5.12 × 10²3.57 × 10³ ± 2.37 × 10³ control)

[0058] The results show that both combinations of oligomers used asprimers performed substantially equally in the assay to amplifyparvovirus B19 sequences. In both assay formats, positive signals weredetected for all of the samples containing 250 or more copies ofparvovirus B19, and positive signals were detected for 70 to 80% of thesamples containing 100 copies.

[0059] In similar experiments, the assay was performed using othercombinations of primers: a promoter primer of SEQ ID NO:3 and SEQ IDNO:13, or a promoter primer of SEQ ID NO:23 and SEQ ID NO:13. Theparvovirus B19-specific portions of these promoter primers are,respectively, SEQ ID NO:4 (CTAGGTTCTGCATGACTGCTACTGGA) and SEQ ID NO:24(see above). The tested samples either contained no parvovirus B19(negative controls) or 1,000 copies of parvovirus B19 per reaction. Tenreplicate samples were tested for each condition. For the combination ofprimers of SEQ ID NO:3 and SEQ ID NO:13, the assay produced a mean RLUsignal of 2,678,709±507,209 for positive samples and 1,841±128 for thenegative controls. For the combination of primers of SEQ ID NO:23 andSEQ ID NO:13, the assay produced a mean RLU signal of 4,074,989±123,420for positive samples and 3,462±2,936 for the negative controls. Thus, anadditional combination of amplification oligomers can be used to performthe assay to detect the presence of parvovirus B19 DNA in a sample.

EXAMPLE 7 Parvovirus B19 Detection Assays Performed with DifferentDetection Probes

[0060] In this example, parvovirus B19 was assayed by usingsubstantially the method described in Example 5. Briefly, samples wereprepared using human plasma that contains no parvovirus (negativecontrol), by adding known amounts of parvovirus B19 (to achieve finalconcentrations of 10,000, 5,000, 1,000, 500, 250, 100, 50, and 25 copiesper ml). In addition to these, previously tested known samplescontaining 1,000 copies of parvovirus B19 per ml were included in thetests as positive controls. Samples were assayed by, first, capturingthe parvovirus B19 DNA from a 1 ml sample by hybridization to acomplementary oligomer (SEQ ID NO:1) which was then hybridized via its3′ poly(A) tail to a complementary poly(T)-oligomer attached to magneticbeads using procedures substantially as described previously (U.S. Pat.No. 6,110,678). Then, a target portion of the parvovirus B19 genomicsequence was amplified in a one-hour TMA reaction that included apromoter primer of SEQ ID NO:23 (comprising the target-specific sequenceof SEQ ID NO:24 and a T7 RNA polymerase promoter sequence of SEQ IDNO:19) and a primer of SEQ ID NO:13. The amplification products weredetected by using detection probes labeled with 2-methyl-AE in areaction to detect relative light units (RLU) as described in detailpreviously (U.S. Pat. Nos. 5,585,481 and 5,639,604). The detectionprobes were synthesized by using standard chemical methods to produceoligomers with a 2′-O-methoxy backbone and having the nucleotidesequences of SEQ ID NO:17(label between nt 7 and 8), SEQ ID NO:27 (labelbetween nt 5 and 6), and SEQ ID NO:28 (label between nt 9 and 10). Twoseparate sets of assays were performed, one in which the detectionresults were obtained by using SEQ ID Nos. 17 and 27, and another inwhich detection results were obtained by using SEQ ID Nos. 17 and 28(using 1×10⁶ RLU per reaction in both sets of assays). Ten replicatesamples were assayed for each of the experimental conditions, and fivereplicate samples were assayed for the positive (1,000 copies) andnegative (0 copies) controls and the NIBSC standard (1,000 genomeequivalents/ml). The results of these tests (detected RLU mean±standarddeviation) are shown in the table below. TABLE 6 Detection of AmplifiedParvovirus B19 Target Sequences Using Different Detection ProbesParvovirus B19 copies/ml SEQ ID NO:17 Probe SEQ ID NO:27 Probe SEQ IDNO:28 Probe 1,000 (positive 273,238 ± 8,370 232,836 ± 7,843 — control)269,226 ± 9,517 — 237,152 ± 7,482 10,000 282,473 ± 6,037 258,127 ±16,557 — 288,209 ± 5,299 — 242,686 ± 13,025 5,000 283,015 ± 3,716245,047 ± 5,292 — 282,135 ± 14,676 — 238,992 ± 3,790 1,000 263,795 ±22,793 241,110 ± 7,211 — 261,161 ± 13,038 — 236,014 ± 6,581 500 224,858± 83,219 216,921 ± 44,803 — 228,023 ± 50,281 — 209,931 ± 49,130 250167,216 ± 80,594 138,291 ± 53,137 — 158,861 ± 100,765 — 144,024 ± 75,550100 84,296 ± 77,843 79,058 ± 70,042 — 97,111 ± 97,430 — 56,746 ± 42,13350 39,551 ± 49,759 30,533 ± 47,622 — 58,045 ± 83,433 — 85,278 ± 101,65225 16,403 ± 44,038 1,526 ± 1,700 — 41,375 ± 72,116 — 57,283 ± 91,578 0(negative control) 819 ± 232 518 ± 55 — 786 ± 66 — 512 ± 29 NIBSCStandard 157,522 ± 67,888 97,318 ± 47,119 — 199,789 ± 68,636 — 191,507 ±51,276

[0061] The results showed that the three assay formats that useddifferent detection probes were substantially equivalent in theirreactivity and sensitivity. That is, based on a positive signal of30,000 or more detected RLU, all three formats detected 100 copies ormore of parvovirus B19 per ml of sample, and frequently detected fewercopies of parvovirus b19 (25 and/or 50 copies/ml).

EXAMPLE 8 Detection of Amplified Parvovirus B19 Sequences UsingDifferent Detection Probes

[0062] This example tested the sensitivity of the assay using individualdetection probes or a mixture of two different detection probes. Themixture of detection probes contained equivalent amounts of probes ofSEQ ID NO:27 and SEQ ID NO:28. Assays compared the detection probemixture to use of either detection probe alone. The assays wereperformed substantially as described in Example 5, but using plasmasamples that contained no parvovirus B19 (negative control), orcontained 500, 250, 100, 50, or 25 copies of parvovirus B19 per ml;positive controls contained 1,000 copies/ml. Samples (1 ml) were assayedby first capturing the parvovirus B19 DNA in a hybridization complex onmagnetic particles by using an oligomer having SEQ ID NO:1 with a 3′poly-A tail, as described above. Then, the parvovirus B19 targetsequence was amplified by using a one-hour TMA reaction that included apromoter primer of SEQ ID NO:23 and a primer of SEQ ID NO:13. Theamplification products were detected by using detection probes of eitherSEQ ID NO:27 or SEQ ID NO:28 individually, or a mixture of probes of SEQID NO:27 and SEQ ID NO:28. The probes were labeled with 2-methyl-AE(between nt 5 and 6 for SEQ ID NO:27, and nt 9/10 for SEQ ID NO:28) andused at an activity of 1×10⁶ RLU per reaction for each probe. For thepositive and negative controls, five replicate samples were tested,whereas for each of the other experimental conditions, twenty replicatesamples were tested. The results (RLU mean±standard deviation) are shownbelow. TABLE 7 Detection Results Using Labeled Probes Alone or a Mixtureof Labeled Probes Parvovirus B19 SEQ ID NO:27 copies/ml SEQ ID NO:27 SEQID NO:28 and SEQ ID NO:28 1,000 464,906 ± 10,157  476,397 ± 8,369 828,543 ± 31,127  (positive control) 500 412,338 ± 95,435  457,938 ±34,499  679,652 ± 210,438 250 296,947 ± 179,021 397,804 ± 111,879554,294 ± 272,640 100 167,560 ± 153,175 262,557 ± 189,262 376,880 ±254,070 50  95,581 ± 145,780  70,364 ± 140,050  82,840 ± 145,301 0 1,046± 205   826 ± 181 1,051 ± 116   (negative control)

[0063] The results show that both of the probes alone and in a mixturedetected parvovirus B19 at 500 copies/ml in all of the assays performed.Samples containing fewer copies of parvovirus B19 were also detected (90to 100% for 250 copies/ml, 75 to 85% for 100 copies/ml, and 30 to 50%for 50 copies/ml). The sensitivities of the three assay formats weresubstantially equivalent.

EXAMPLE 9 Parvovirus B19 Detection Using Different Detection Probes

[0064] In this example, the amplified products produced using the methodsubstantially as described in Example 8 were detected using variousdetection probe oligomers. The detection probe oligomers varied from oneanother either in their nucleotide sequence or, for probe oligomers withthe same nucleotide sequence, at the position of label attachment to theoligomer. All of the probes were synthesized in vitro using standardchemical methods to produce an oligomer of specified sequence with a2′-O-methoxy backbone. Oligomers were labeled with 2-methyl-AE aspreviously described (U.S. Pat. Nos. 5,585,481 and 5,639,604) using alinker compound to attach the label compound to the oligomer and used atan activity of 1×10⁶ RLU per reaction. The label position on theoligomer is referred to by the adjacent nucleotide positions, e.g.,“12/13” means that the linker and attached label are located between nt12 and nt 13 of the oligomer. The detection probe oligomers tested inthese experiments are summarized below. TABLE 8 Labeled Probes SEQ ID NONucleotide Sequence Label Positions 27 GTCATGGACAGTTATCTGAC 7/8, 9/10,12/13, and 13/14 28 GTATTATCTAGTGAAGACTTAC 12/13 30CTAGTGAAGACTTACACAAGC 5/6 and 13/14 31 GTGAAGACTTACACAAGCCTG 9/10 and10/11 32 GCAGTATTATCTAGTGAAGAC 8/9 and 12/13 34 CAAAGTCATGGACAGTTATCTG7/8, 9/10, 11/12, 13/14, 16/17, and 17/18 36 CTGTTTGACTTAGTTGCTCG 6/7,7/8, 10/11, 11/12, 14/15, and 15/16 37 CTCTCCAGACTTATATAGTCATCAT 7/8,8/9, 9/10, 11/12, 12/13, 14/15, 16/17, 17/18, and 18/19

[0065] The results of assays that used these detection probes are shownbelow, reported as the average (mean) RLU detected. Each probe wastested in five replicate assays of human plasma samples that containedno parvovirus B19 DNA (negative samples) and plasma that contained 1,000copies/ml of parvovirus B19 (positive samples). The ratio of RLUdetected in the positive samples to RLU detected in the negative samples(detection ratio) was determined using the average RLU results for eachprobe. TABLE 9 Results Obtained By Using Different Labeled Probes SEQ IDNO. and Positive Samples Negative Samples Detection Label Position (meanRLU) (mean RLU) Ratio NO:27, Label 7/8 287,160 409 702 NO:27, Label 9/10419,399 610 687 NO:27, Label 12/13 415,421 691 601 NO:27, Label 13/14461,686 747 618 NO:28, Label 12/13 383,934 864 444 NO:30, Label 5/6432,460 874 495 NO:30, Label 13/14 422,976 2,626 161 NO:31, Label 9/10413,436 3,107 133 NO:31, Label 10/11 545,659 3,398 160 NO:32, Label 8/9471,379 864 545 NO:32, Label 12/13 445,970 473 943 NO:34, Label 7/8535,343 7,105 75 NO:34, Label 9/10 473,386 1,044 453 NO:34, Label 11/12369,158 647 570 NO:34, Label 13/14 364,239 823 442 NO:34, Label 16/17220,368 672 328 NO:34, Label 17/18 373,932 950 393 NO:36, Label 6/7520,799 814 639 NO:36, Label 7/8 482,847 792 609 NO:36, Label 10/11370,929 633 586 NQ:36, Label 11/12 343,754 757 454 NO:36, Label 14/15364,239 823 442 NO:36, Label 15/16 382,139 1,016 376 NO:37, Label 7/8336,293 61,020 5 NO:37, Label 8/9 81,986 1,314 62 NO:37, Label 9/10516,495 57,853 9 NO:37, Label 11/12 559,173 133,530 4 NO:37, Label 12/13506,083 121,133 4 NO:37, Label 14/15 593,889 54,116 5 NO:37, Label 16/17439,755 94,380 4 NO:37, Label 17/18 361,001 91,163 4 NO:37, Label 18/19222,039 2,233 99

[0066] The results showed that a variety of different detection probesmay be used to detect parvovirus B19 sequences in the amplificationproduct because all of the probes tested produced at least four-foldmore signal than the negative controls. Preferred embodiments generallyhave a detection ratio of 10 or greater. More preferably, the detectionratio is 100 or greater, and most preferably is in a range of 300 to950. These results also showed that, for the same nucleotide sequence,the position of the label on the oligomer may influence the detectionsignal produced.

[0067] The present invention has been described in the context ofparticular examples and preferred embodiments. Those skilled in the artwill appreciate that other embodiments are encompassed within theinvention defined by the claims that follow.

1 41 1 28 DNA Artificial Sequence Description of Artificial Sequencesynthetic construct 1 gttggctata cctaaagtca tgaatcct 28 2 32 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 2 gccagttggc tatacctaaa gtcatgaatc ct 32 3 53 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 3aatttaatac gactcactat agggagacta ggttctgcat gactgctact gga 53 4 26 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 4 ctaggttctg catgactgct actgga 26 5 53 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 5 aatttaatacgactcactat agggagactg catgactgct actggatgat aag 53 6 26 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 6ctgcatgact gctactggat gataag 26 7 56 DNA Artificial Sequence Descriptionof Artificial Sequence synthetic construct 7 aatttaatac gactcactatagggagacta ggttctgcat gactgctact ggatga 56 8 29 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 8 ctaggttctgcatgactgct actggatga 29 9 52 DNA Artificial Sequence Description ofArtificial Sequence synthetic construct 9 aatttaatac gactcactatagggagagtt ctgcatgact gctactggat ga 52 10 25 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 10 gttctgcatgactgctactg gatga 25 11 53 DNA Artificial Sequence Description ofArtificial Sequence synthetic construct 11 aatttaatac gactcactatagggagattc tcctctaggt tctgcatgac tgc 53 12 26 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 12 ttctcctctaggttctgcat gactgc 26 13 21 DNA Artificial Sequence Description ofArtificial Sequence synthetic construct 13 cccctagaaa acccatcctc t 21 1429 DNA Artificial Sequence Description of Artificial Sequence syntheticconstruct 14 ctctccagac ttatatagtc atcattttc 29 15 25 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 15ctctccagac ttatatagtc atcat 25 16 23 DNA Artificial Sequence Descriptionof Artificial Sequence synthetic construct 16 atcccctaga aaacccatcc tct23 17 25 DNA Artificial Sequence Description of Artificial Sequencesynthetic construct 17 gacagttatc tgaccacccc catgc 25 18 22 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 18 catggacagt tatctgacca cc 22 19 27 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 19 aatttaatacgactcactat agggaga 27 20 27 DNA Artificial Sequence Description ofArtificial Sequence synthetic construct 20 catcactttc ccaccatttg ccacttt27 21 32 DNA Artificial Sequence Description of Artificial Sequencesynthetic construct 21 gcaaatttat catcactttc ccaccatttg cc 32 22 28 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 22 aggattcatg actttaggta tagccaac 28 23 52 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 23aatttaatac gactcactat agggagaagt accgggtagt tgtacgctaa ct 52 24 25 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 24 agtaccgggt agttgtacgc taact 25 25 53 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 25aatttaatac gactcactat agggagacta ggttctgcat gactgctact gga 53 26 26 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 26 ctaggttctg catgactgct actgga 26 27 20 DNA ArtificialSequence Description of Artificial Sequence synthetic construct 27gtcatggaca gttatctgac 20 28 22 DNA Artificial Sequence Description ofArtificial Sequence synthetic construct 28 gtattatcta gtgaagactt ac 2229 37 DNA Artificial Sequence Description of Artificial Sequencesynthetic construct 29 aaagtggcaa atggtgggaa agtgatgata aatttgc 37 30 21DNA Artificial Sequence Description of Artificial Sequence syntheticconstruct 30 ctagtgaaga cttacacaag c 21 31 21 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 31 gtgaagacttacacaagcct g 21 32 21 DNA Artificial Sequence Description of ArtificialSequence synthetic construct 32 gcagtattat ctagtgaaga c 21 33 34 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 33 gcagtattat ctagtgaaga cttacacaag cctg 34 34 22 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 34 caaagtcatg gacagttatc tg 22 35 35 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 35 caaagtcatggacagttatc tgaccacccc catgc 35 36 20 DNA Artificial Sequence Descriptionof Artificial Sequence synthetic construct 36 ctgtttgact tagttgctcg 2037 25 DNA Artificial Sequence Description of Artificial Sequencesynthetic construct 37 ctctccagac ttatatagtc atcat 25 38 18 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 38 gtcatggaca gttatctg 18 39 18 DNA Artificial SequenceDescription of Artificial Sequence synthetic construct 39 gtgaagacttacacaagc 18 40 18 DNA Artificial Sequence Description of ArtificialSequence synthetic construct 40 gtattatcta gtgaagac 18 41 22 DNAArtificial Sequence Description of Artificial Sequence syntheticconstruct 41 catcactttc ccaccatttg cc 22

We claim:
 1. A combination of at least two separate nucleic acidoligomers, wherein the oligomers are selected from the group consistingof: SEQ ID NO:4, a complementary sequence, or RNA equivalent thereof,optionally including a promoter sequence joined to a 5′ terminus of thesequence, SEQ ID NO:6, a complementary sequence, or RNA equivalentthereof, optionally including a promoter sequence joined to a 5′terminus of the sequence, SEQ ID NO:8, a complementary sequence, or RNAequivalent thereof, optionally including a promoter sequence joined to a5′ terminus of the sequence, SEQ ID NO:10, a complementary sequence, orRNA equivalent thereof, optionally including a promoter sequence joinedto a 5′ terminus of the sequence, SEQ ID NO:12, a complementarysequence, or RNA equivalent thereof, optionally including a promotersequence joined to a 5′ terminus of the sequence, SEQ ID NO:24, acomplementary sequence, or RNA equivalent thereof, optionally includinga promoter sequence joined to a 5′ terminus of the sequence, SEQ IDNO:26, a complementary sequence, or RNA equivalent thereof, optionallyincluding a promoter sequence joined to a 5′ terminus of the sequence,SEQ ID NO:13, a complementary sequence, or RNA equivalent thereof, SEQID NO:14, a complementary sequence, or RNA equivalent thereof, SEQ IDNO:15, a complementary sequence, or RNA equivalent thereof, SEQ IDNO:16, a complementary sequence, or RNA equivalent thereof, SEQ IDNO:17, a complementary sequence, or RNA equivalent thereof, optionallywith a detectable label joined to the sequence, SEQ ID NO:18, acomplementary sequence, or RNA equivalent thereof, optionally with adetectable label joined to the sequence, SEQ ID NO:27, a complementarysequence, or RNA equivalent thereof, optionally with a detectable labeljoined to the sequence, SEQ ID NO:28, a complementary sequence, or RNAequivalent thereof, optionally with a detectable label joined to thesequence, SEQ ID NO:30, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence, SEQID NO:31, a complementary sequence, or RNA equivalent thereof,optionally with a detectable label joined to the sequence, SEQ ID NO:32,a complementary sequence, or RNA equivalent thereof, optionally with adetectable label joined to the sequence, SEQ ID NO:34, a complementarysequence, or RNA equivalent thereof, optionally with a detectable labeljoined to the sequence, SEQ ID NO:36, a complementary sequence, or RNAequivalent thereof, optionally with a detectable label joined to thesequence, SEQ ID NO:37, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence, SEQID NO:1, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence, SEQ ID NO:2, a complementary sequence, or RNA equivalentthereof, and optionally a 3′ tail portion that is nonspecific for aparvovirus target sequence, SEQ ID NO:20, a complementary sequence, orRNA equivalent thereof, and optionally a 3′ tail portion that isnonspecific for a parvovirus target sequence, and SEQ ID NO:21, acomplementary sequence, or RNA equivalent thereof, and optionally a 3′tail portion that is nonspecific for a parvovirus target sequence.
 2. Acombination of oligomers of claim 1, selected from the group consistingof: SEQ ID NO:13, a complementary sequence, or RNA equivalent thereof,SEQ ID NO:24, a complementary sequence, or RNA equivalent thereof,optionally including a promoter sequence joined to the 5′ terminus ofthe sequence, SEQ ID NO:27, a complementary sequence, or RNA equivalentthereof, optionally with a detectable label joined to the sequence, andSEQ ID NO:28, a complementary sequence, or RNA equivalent thereof,optionally with a detectable label joined to the sequence.
 3. Acombination of oligomers of claim 1, wherein the promoter sequencejoined to the 5′ terminus of the sequence is the promoter sequence ofSEQ ID NO:19.
 4. A nucleic acid oligomer consisting of a target-specificsequence contained in SEQ ID NO:29, a complementary sequence, or RNAequivalent thereof, and optionally a 3′ tail portion that is nonspecificfor a parvovirus target sequence.
 5. A nucleic acid oligomer of claim 4,wherein the oligomer contains the target-specific sequence of SEQ IDNO:20, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence.
 6. A nucleic acid oligomer of claim 4, wherein the oligomercontains the target-specific sequence of SEQ ID NO:21, a complementarysequence, or RNA equivalent thereof, and optionally a 3′ tail portionthat is nonspecific for a parvovirus target sequence.
 7. A nucleic acidoligomer of claim 4, wherein the oligomer contains the target-specificsequence of SEQ ID NO:41, a complementary sequence, or RNA equivalentthereof, and optionally a 3′ tail portion that is nonspecific for aparvovirus target sequence.
 8. A nucleic acid oligomer consisting of atarget-specific sequence of at least 25 contiguous bases contained inSEQ ID NO:1, a complementary sequence, or RNA equivalent thereof, andoptionally a 3′ tail portion that is nonspecific for a parvovirus targetsequence.
 9. A nucleic acid oligomer consisting of a sequence containedin SEQ ID NO:33, a complementary sequence, or RNA equivalent thereof.10. A nucleic acid oliogmer of claim 9, wherein the oligomer comprises asequence consisting of SEQ ID NO:40, a complementary sequence, or RNAequivalent thereof.
 11. A nucleic acid oliogmer of claim 9, wherein theoligomer contains the sequence of SEQ ID NO:32 or SEQ ID NO:28, acomplementary sequence, or RNA equivalent thereof.
 12. A nucleic acidoliogmer of claim 9, wherein the oligomer comprises a sequenceconsisting of SEQ ID NO:39, a complementary sequence, or RNA equivalentthereof.
 13. A nucleic acid oliogmer of claim 9, wherein the oligomercontains the sequence of SEQ ID NO:30 or SEQ ID NO:31, a complementarysequence, or RNA equivalent thereof.
 14. A nucleic acid oliogmer ofclaim 9, wherein the oligomer has a backbone that includes at least one2′-O-methoxy linkage.
 15. A nucleic acid oligomer consisting of asequence contained in SEQ ID NO:35, a complementary sequence, or RNAequivalent thereof.
 16. A nucleic acid oligomer of claim 15, wherein theoligomer contains the sequence of SEQ ID NO:17, SEQ ID NO:27, or SEQ IDNO:34, a complementary sequence, or RNA equivalent thereof.
 17. Anucleic acid oligomer of claim 15, wherein the oligomer has a backbonethat includes at least one 2′-O-methoxy linkage.
 18. A nucleic acidoligomer selected from the group consisting of: SEQ ID NO:36, acomplementary sequence, or RNA equivalent thereof, and SEQ ID NO:37, acomplementary sequence, or RNA equivalent thereof.
 19. A nucleic acidoligomer of claim 18, wherein the oligomer has a backbone that includesat least one 2′-O-methoxy linkage.
 20. A method of detecting humanparvovirus B19 nucleic acid in a biological sample, comprising the stepsof: providing a biological sample containing parvovirus B19 nucleicacid; amplifying in vitro a portion of the parvovirus B19 nucleic acidby using at least one nucleic acid polymerase activity and at least onefirst amplification oligomer and one second amplification oligomer,selected from the group consisting of first amplification oligomers ofSEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:24, or SEQ ID NO:26, each optionally including a promoter sequencejoined to a 5′ terminus of the sequence, and second amplificationoligomers of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16;and detecting an amplified product of the parvovirus B19 nucleic acid byusing a labeled detection probe that hybridizes specifically with theamplified product, thereby indicating presence of parvovirus B19 nucleicacid in the biological sample.
 21. The method of claim 20, wherein theamplifying step uses at least two oligomers selected from the followinggroup of oligomer pairs: SEQ ID NO:4 including a promoter sequencejoined to the 5′ terminus, with SEQ ID NO:13, SEQ ID NO:6 including apromoter sequence joined to the 5′ terminus, with SEQ ID NO:13, SEQ IDNO:24 including a promoter sequence joined to the 5′ terminus, with SEQID NO:13, and SEQ ID NO:26 including a promoter sequence joined to the5′ terminus, with SEQ ID NO:13.
 22. The method of claim 20, wherein theamplifying step uses an amplification reaction that is substantiallyisothermal.
 23. The method of claim 20, wherein the detecting step usesa labeled detection probe selected from the group consisting of SEQ IDNO:17, SEQ ID NO!18, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:37. 24.The method of claim 20, wherein the detecting step uses a labeleddetection probe selected from the group consisting of SEQ ID NO:17, SEQID NO:27, and SEQ ID NO:28.
 25. The method of claim 20, wherein thedetecting step uses a labeled detection probe comprising a backbone thatincludes at least one 2′-O-methoxy linkage.
 26. The method of claim 20,wherein the detecting step uses a labeled detection probe that includesa label that is detected in a homogeneous reaction.
 27. The method ofclaim 20, wherein the detecting step uses a labeled detection probe thatincludes a chemiluminescent label attached to the oligomer via a linkercompound.
 28. The method of claim 20, further comprising the steps of:contacting the biological sample with at least one capture oligomercomprising a target-specific sequence that hybridizes to a parvovirusB19 target sequence, thus forming a complex comprising the captureoligomer and parvovirus B19 nucleic acid; and separating the complexfrom the biological sample before the amplifying step.
 29. The method ofclaim 28, wherein the capture oligomer comprises at least 25 contiguousbases of a target-specific sequence contained in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:20, or SEQ ID NO:21, or RNA equivalents thereof.
 30. Themethod of claim 28, wherein the capture oligomer comprises a backbonethat includes at least one 2′-O-methoxy linkage.